Project Summary This project will explore the mechanochemistry of myosin II filaments moving on actin filaments using in vitro methods. We will use our recently-developed inverted motility assay to measure myosin filament velocities moving on actin filaments. By varying the number of myosin heads in the filaments and the ATP concentration, we have developed a deterministic model describing the parameters that define and limit the rate of filament- filament sliding that inform mechanisms of muscle contraction. Our prior work revealed how assembling myosin into filaments allows for attachment-limited kinetics at physiological numbers of myosins in the filaments, in contrast to the current paradigm in the field of detachment-limited kinetics. We use this assay as the basis for addressing further specific structural and kinetic hypotheses about how force and motion are generated when myosin is incorporated into a filament. Myosin filaments, both reconstituted and native, from smooth, skeletal, insect flight, and cardiac muscle will be compared. Genetically engineered fruit flies will be generated to express insect flight muscle myosin with either shortened or lengthened S2 domains, which is the myosin heavy chain domain that we hypothesize to underlie the attachment-limited kinetics mentioned above. The effects of phosphorylation of the myosin regulatory light chains will be examined, with the goal of testing a novel hypothesis about the activity of myosin with only one of its heads phosphorylated. The assay will be modified in two ways to extend its utility. First, single myosin heads will be labeled with quantum dots an incorporated into co-filaments. The global motion of the moving filament and the quantum dot within the moving filament will simultaneously visualized, and by analysis of the motion by tracking software, the presence or absence of predicted mechanical signatures will be assessed. The identity of those signatures will be assessed by correlation to changing experimental conditions that we predict will change the signature. Also, the inverted in vitro motility assay will be modified to allow measurement of myosin filament moving under load, allowing underlying effects of load on kinetics to be examined and allow comparisons of the relative ability of different myosins to generate power. Final, we will build on these approaches by using the inverted motility assay to probe mechanisms underlying the regulation of thin filament ?thick filament sliding by calcium and myosin head binding.